Sucrose phosphorylase (hereinafter, also referred to as SP or SP enzyme) is an enzyme utilized for example in α-glucan synthesis and glucose-1-phosphate (G-1-P) synthesis.
α-Glucan (particularly insoluble amylose) is expected to have the same functions as those of dietary fibers and can be expected to be useful in health food. Further, an amylose that is a linear α-glucan has characteristics such as being capable of including, for example, iodine, fatty acids, and the like in its molecule, and can thus be expected to be useful in fields such as pharmaceuticals, cosmetics, and sanitary products. An amylose can also be utilized as a crude material for producing cyclodextrin and cycloamylose having an inclusion ability similar to that of amylose. Further, a film containing an amylose has tensile strength comparable to general-purpose plastics and is has good prospects as a raw material for biodegradable plastics. Thus, amylose is expected to be useful in various applications. However, substantially pure amylose is rarely obtained, is very expensive and is thus distributed only at the reagent level and is scarcely utilized as an industrial raw material. Accordingly, there is demand for a method of producing an amylose stably and inexpensively.
G-1-P is utilized, for example, as a medical antibacterial agent, an anti-tumor agent (as a platinum complex), as a drug for treating heart disease (as an amine salt) and a substrate for α-glucan synthesis.
SPs are reported to exist in some bacteria (bacteria of the genus Streptococcus, microorganisms of the genus Leuconostoc, Escherichia coli, lactic acid bacteria, and the like), and the amino acid sequences and base sequences of SPs found in these bacteria are known.
Various SPs can be used in the production of an α-glucan or G-1-P, and SP derived from bacteria of the genus Leuconostoc is often used. This is because a relatively large amount of the enzyme can be easily obtained.
In industrial production of an α-glucan, it is necessary to essentially remove other contaminating enzyme activities, particularly phosphatase activity and amylase activity. This is because if phosphatase is present during the synthesis of an α-glucan by SP and GP, G-1-P synthesized as a reaction intermediate is degraded to reduce the yield of the α-glucan. This is because if amylase is present, synthesized α-glucan is degraded to reduce the molecular weight thereof. Accordingly, it is necessary to remove these contaminating enzymes. Escherichia coli and Bacillus subtilis are desirable hosts for expressing an SP gene when producing a large amount of SP. However, as shown in FIGS. 2 and 3, Escherichia coli has amylase activity and phosphatase activity, and Bacillus subtilis has amylase activity. However, as shown in FIGS. 2 and 3, the enzymes possessed by these hosts cannot be inactivated by heat treatment at 55° C., but can be almost completely inactivated by heat treatment at a temperature of 60° C. Therefore, an SP having thermostability whereby its activity is not lost, even after heat treatment at 60° C., or an SP having higher thermostability than that of amylase or phosphatase has been desired.
For reference, specific numerical values of the amylase activity and phosphatase activity in cell extracts from various bacteria (E. coli TG-1, E. coli BL21, and Bacillus subtilis ANA-1) before and after heating are shown in the following Table 1.
TABLE 1Phosphatase activity (%)Amylase activity (%)TG-1BL21TG-1BL21ANA-1Before100100100100100heating50° C.99.198.621.628.633.855° C.60.974.59.19.719.860° C.2.93.10.403.065° C.2.52.00.902.4
However, an SP which has thermostability, particularly an SP which can maintain sufficient activity at high temperatures (for example 60° C. to 75° C.), is not known.
In production of an α-glucan or G-1-P, it is also preferable to conduct the reaction at temperatures as high as possible. This is because when the enzyme reaction is carried out at high temperatures, the reaction rate is generally increased and the operability of an α-glucan is improved. An α-Glucan, particularly an amylose, is aged and insolubilized to form precipitates or gel. This aging rate is well-known to depend on temperature. When the reaction temperature is low, there arise problems in operability at a later stage, such as gelation of the amylose solution after production. For carrying out the reaction at high temperatures, it is necessary that the enzyme be thermostable.
However, all of the bacteria described above are mesophilic bacteria, and an SP having thermostability which is derived from a bacteria, particularly an SP which can maintain sufficient activity at high temperatures (for example, 50° C. to 60° C.), is not known. Therefore, in the case of conventional SP, purification costs cannot be reduced by heating treatment, and the reaction cannot be carried out at high temperature either.
With respect to an SP derived from organisms other than bacteria (prokaryotes), an SP derived from mold (eukaryotes) is reported (see non-patent document 1). According to this reference, an SP derived from Monilia sitophila (also known as Neurospora intermedia) retains 90% or more of its activity after heating at 70° C. for 30 minutes.
However, the experimental results reported in this reference are obtained by using an SP of very low purity, so doubts remain as to whether the observed SP activity can be truly quantitatively measured SP activity. Further, many molds secrete amylase to the outside of the bacterial body, so it is necessary to highly purify the SP enzyme for use, and purification of the SP enzyme requires significant time and costs. It is impossible to introduce this mold-derived SP enzyme into a host secreting phosphatase and amylase in a lower amount using genetic recombination technology. This is because neither the amino acid sequence nor the base sequence of this mold-derived SP is known.
SP enzyme is reported to show an improvement in it's thermostability by immobilizing the enzyme, but the immobilized enzyme has disadvantages such as changes in substrate specificity, and there is a limit to the improvement of thermostability by immobilization, so it is desired to improve the thermostability of the SP enzyme itself. There are no reports of improving the thermostability of an SP enzyme by introduction of a mutation.
The apparent thermostability of SP enzyme is improved in the presence of sucrose at high concentrations (see Patent Document 1). However, there is a limit to the increase in thermostability achieved in the presence of sucrose at high concentrations, so it is desired to improve the thermostability of the SP enzyme itself.
In order to solve these problems, an SP which is advantageous for industrial utilization and has high thermostability is required.
Theoretical methods for making a general enzyme more thermostable, such as proline theory and amino acid substitution based on enzyme steric structure information have been tried, but have not necessarily succeeded. For this reason, methods based upon random mutation, or methods using a combination of random mutation and theoretical methodology is currently being carried out. However, in any of these methods, every protein must be characterized by trial and error.
Regarding enzymes other than SP, it has been reported that, once the position of a particular amino acid(s) involved in improving the thermostability of an enzyme is determined, an enzyme can be made thermostable by substitution of the specified amino acid residue(s) with other amino acid residues (for example see Non-Patent Documents 2 to 4).
(Patent Document 1)
International Publication No. 02/097077 Pamphlet
(Non-Patent Document 1)
M. C. B. Pimentel et al., “SCREENING, THERMAL PROPERTIES AND PRODUCTION IN YAM EXTRACT OF FUNGAL SUCROSE PHOSPHORYLASE”, Rev. Microbiol., Sao Paulo, 1992, 23(3), pp. 199-205
(Non-Patent Document 2)
Martin Lehmann and Markus Wyss: “Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution”, Current Opinion in Biotechnology, 2001, 12, pp. 371-375
(Non-Patent Document 3)
M. Lehmann et al., “The consensus concept for thermostability engineering of proteins”, Biochemica Biophysica Acta, 2000, 1543, pp. 408-415
(Non-Patent Document 4)
Junichi Miyazaki et al., “Ancestral Residues Stabilizing 3-Isopropylmalate Dehydrogenase of an Extreme Thermophile: Experimental Evidence Supporting the Thermophilic Common Ancestor Hypothesis”, J. Biochem., 2001, 129, pp. 777-782